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Systematic Production of Inactivating and Non-Inactivating Suppressor Mutations at the relA Locus That Compensate the Detrimental Effects of Complete spoT Loss and Affect Glycogen Content in Escherichia coli

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dc.contributor.author Montero, Manuel
dc.contributor.author Rahimpour, Mehdi
dc.contributor.author Viale, Alejandro M.
dc.contributor.author Almagro, Goizeder
dc.contributor.author Eydallin, Gustavo
dc.contributor.author Sevilla, Angel
dc.contributor.author Canovas, Manuel
dc.contributor.author Bernal, Cristina
dc.contributor.author Lozano, Ana Belen
dc.contributor.author Munoz, Francisco Jose
dc.contributor.author Baroja-Fernandez, Edurne
dc.contributor.author Bahaji, Abdellatif
dc.contributor.author Mori, Hirotada
dc.contributor.author Codoner, Francisco M.
dc.contributor.author Pozueta-Romero, Javier
dc.date.accessioned 2016-08-19T07:43:52Z
dc.date.available 2016-08-19T07:43:52Z
dc.date.issued 2014-09-04
dc.identifier.issn 1932-6203
dc.identifier.uri http://hdl.handle.net/10061/10978
dc.description.abstract In Escherichia coli, ppGpp is a major determinant of growth and glycogen accumulation. Levels of this signaling nucleotide are controlled by the balanced activities of the ppGpp RelA synthetase and the dual-function hydrolase/synthetase SpoT. Here we report the construction of spoT null (ΔspoT) mutants obtained by transducing a ΔspoT allele from ΔrelAΔspoT double mutants into relA+ cells. Iodine staining of randomly selected transductants cultured on a rich complex medium revealed differences in glycogen content among them. Sequence and biochemical analyses of 8 ΔspoT clones displaying glycogen-deficient phenotypes revealed different inactivating mutations in relA and no detectable ppGpp when cells were cultured on a rich complex medium. Remarkably, although the co-existence of ΔspoT with relA proficient alleles has generally been considered synthetically lethal, we found that 11 ΔspoT clones displaying high glycogen phenotypes possessed relA mutant alleles with non-inactivating mutations that encoded stable RelA proteins and ppGpp contents reaching 45-85% of those of wild type cells. None of the ΔspoT clones, however, could grow on M9-glucose minimal medium. Both Sanger sequencing of specific genes and high-throughput genome sequencing of the ΔspoT clones revealed that suppressor mutations were restricted to the relA locus. The overall results (a) defined in around 4 nmoles ppGpp/g dry weight the threshold cellular levels that suffice to trigger net glycogen accumulation, (b) showed that mutations in relA, but not necessarily inactivating mutations, can be selected to compensate total SpoT function(s) loss, and (c) provided useful tools for studies of the in vivo regulation of E. coli RelA ppGpp synthetase.
dc.language.iso en
dc.publisher Public Library of Science
dc.rights c 2014 Montero et al.
dc.title Systematic Production of Inactivating and Non-Inactivating Suppressor Mutations at the relA Locus That Compensate the Detrimental Effects of Complete spoT Loss and Affect Glycogen Content in Escherichia coli
dc.type.nii Journal Article
dc.contributor.transcription モリ, ヒロタダ
dc.contributor.alternative 森, 浩禎
dc.identifier.fulltexturl http://dx.doi.org/10.1371%2Fjournal.pone.0106938
dc.textversion Publisher
dc.identifier.jtitle PLoS ONE
dc.identifier.volume 9
dc.identifier.issue 9
dc.identifier.spage e106938
dc.relation.doi 10.1371/journal.pone.0106938
dc.identifier.NAIST-ID 73290108
dc.relation.pmid 25188023


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